Formation and role of glycine betaine in the moderate halophile Vibrio costicola

The moderate halophile Vibrio costicola, growing on a chemically-defined medium, transformed choline into glycine betaine (betaine) by the membrane-bound enzyme choline dehydrogenase and the cytoplasmic enzyme betainal (betaine aldehyde) dehydrogenase. Choline dehydrogenase was strongly induced and betainal dehydrogenase less strongly induced by choline. The formation of these enzymes was also regulated by the NaCl concentration of the growth medium, increasing with increasing NaCl concentrations. Intracellular betaine concentrations also increased with increasing choline and NaCl concentrations in the medium. This increase was almost completely blocked by chloramphenicol, which does not block the increase in salt-tolerant active transport on transfer from a low to a high salt concentration.Choline dehydrogenase was inhibited by chloride salts of Na⁺, K⁺, and NH _inf4 ^su+ , the inhibition being due to the Cl^- ions. Betainal dehydrogenase was stimulated by 0.5 M salts and could function in up to 2.0 M salts.Cells grew as well in the presence as in the absence of choline in 0.5 M and 1.0 M NaCl, but formed no intracellular betaine. Choline stimulated growth in 2.0 M NaCl and was essential for growth in 3.0 M NaCl. Thus, while betaine is important for some of the adaptations to high salt concentration by V. costicola, it by no means accounts for all of them.Abbreviations CDMM chemically-defined minimal medium - PPT proteose-peptone tryptone medium - SDS sodium dodecyl sulfate Deceased, 1987     

Glycine Betaine, Carnitine, and Choline Enhance Salinity Tolerance and Prevent the Accumulation of Sodium to a Level Inhibiting Growth of Tetragenococcus halophila

Natural-abundance ¹³C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na⁺ content and confer a much higher salt tolerance to T. halophila.     

Choline Derivatives Involved in Osmotolerance of Penicillium fellutanum

Penicillium fellutanum is osmotolerant and xerotolerant when cultured in a low-phosphate medium containing 3 M NaCl. Glycerol and erythritol accumulated in cultures with NaCl concentrations up to 2 M; glycerol was the only detectable polyol in cultures containing 3 M NaCl. In cultures with 3 M NaCl, the intracellular levels of glycine betaine and choline-O-sulfate were 22- and 2.6-fold greater (70 and 46 mM), respectively, than those of cultures without added NaCl. The levels of glycine betaine and glycerol decreased in mycelia transferred from a medium containing 3 M NaCl into a fresh medium without added NaCl. NaCl at 3 M inhibited mycelial mass accumulation; this inhibition was partially corrected by supplementation of cultures with glycine betaine (2 mM) or choline-O-sulfate (10 mM). The presence of exogenous choline chloride (2 mM) in plate cultures protected the cells from stress from 3 M NaCl. The data suggest that glycine betaine and choline-O-sulfate are secondary osmoprotectants which are effective at the point that the cell is incapable of synthesizing more glycerol.     

Functional Expression of Sinorhizobium meliloti BetS, a High-Affinity Betaine Transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Østerås, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na⁺/H⁺ antiporters in B. japonicum could explain its very high Na⁺ sensitivity.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Osmoregulation in Rhodobacter sphaeroides.

Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light. The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths. The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions. Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized. The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 microM to 2.0 mM). Only one kinetically distinguishable choline transport system could be detected. Kt values of 2.4 and 3.0 microM and maximal rates of choline uptake (Vmax) of 5.4 and 4.2 nmol of choline/min.mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively. Choline transport was not inhibited by a 25-fold excess of L-proline or betaine. Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl. In cells grown and assayed in the absence of NaCl, betaine transport occurred with a Kt of 15.1 microM and a Vmax of 3.2 nmol/min . mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 microM and 9.2 nmol of betaine/min . mg of protein. This system was also able to transport L-proline, but with a lower affinity than that for betaine. The addition of choline of betaine to the growth medium did not result in the induction of additional transport systems.     

Hypersaline stress induces the turnover of phosphatidylcholine and results in the synthesis of the renal osmoprotectant glycerophosphocholine in Saccharomyces cerevisiae

The role of phosphatidylcholine turnover during hypersaline stress is investigated in Saccharomyces cerevisiae. In the wild-type strain, 2180-1A hypersaline stress induced the rapid turnover of phosphatidylcholine, a major membrane lipid. Yeast cells were grown in the presence of [14C]-choline to label phosphatidylcholine. Upon shifting the cells to medium with 0.8 M NaCl, phosphatidylcholine levels were diminished by c. 30% within 20 min to yield glycerophosphocholine, a methylamine osmoprotectant that has been previously identified in renal cells. High-performance liquid chromatography studies showed that osmotically mediated glycerophosphocholine production was enhanced if 10 mM choline was added as a supplement to synthetic dextrose medium with 1.6 M NaCl, but glycine betaine was not detected. Enhanced glycerophosphocholine production also correlated with improved growth in media containing 1.6 M NaCl and choline. Enhanced growth is specific to methylamines: salt-stressed cells supplemented with 10 mM choline or glycine betaine showed enhanced growth relative to unsupplemented control cultures, but other additives had no effect on growth or adversely affected it. Nutritional effects are ruled out because yeast cannot use choline or glycine betaine as carbon or nitrogen sources in normal or high-salt medium. Finally, enhanced growth in hypersaline media with choline or glycine betaine is dependent on the choline permease Hnm1. These results in yeast highlight a similarity with mammalian renal cells, namely that phosphatidylcholine turnover contributes to osmotic adaptation via synthesis of the osmoprotectant glycerophosphocholine.     

Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway.

The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium.     

Choline and Glycine Betaine Uptake in Various Strains of Rhizobia Isolated from Nodules of Vicia faba var. major and Cicer arietinum L.: Modulation by Salt,Choline, and Glycine Betaine

Two strains of Rhizobia isolated from nodules of Vicia faba var. major and one strain isolated from nodules of Cicer arietinum L. were characterized for salt resistance. The presence of 1 mM glycine betaine or choline in a minimal medium with added NaCl had a beneficial role on the growth of the three strains. Both molecules were found to be taken up by cells obtained at low osmolarity, and whereas glycine betaine uptake activity was stimulated significantly in cells grown in the presence of 0.15 M NaCl, choline uptake activity was strongly inhibited by salt in all tested strains. However, in cells grown with exogenous choline, the uptake inhibition exerted by salt was relieved, mainly in the strain isolated from nodules of C. arietinum L. On the basis of kinetics determinations, in control cells as well as in salt-stressed cells, only high-affinity activities were observed for glycine betaine and choline (apparent K _m s between 3 and 18 μM). Periplasmic proteins that bound glycine betaine or choline were identified. In nondenaturing conditions, these proteins extracted from the various strains showed different electrophoretic mobility with always a less negative entire charge than the analogous proteins from Rhizobium meliloti. Received: 29 July 1996 / Accepted: 10 September 1996     

Betaine Transport Imparts Osmotolerance on a Strain of Lactobacillus acidophilus

Unlike most Lactobacillus acidophilus strains, a specific strain, L. acidophilus IFO 3532, was found to grow in rich medium containing 1 M sodium acetate, KCl, or NaCl. This strain could also grow with up to 1.8 M NaCl or 3 M nonelectrolytes (fructose, xylose, or sorbitol) added. Thus, this strain was tolerant to osmotic pressures up to 2.8 osM. A search for an intracellular solute which conferred osmoprotection led to the identification of glycine betaine (betaine). Betaine was accumulated to high concentrations in cells growing in MRS medium supplemented with 1 M KCl or NaCl. Uptake of [¹⁴C]betaine by L. acidophilus 3532 cells suspended in buffer was stimulated by increasing the medium osmotic pressure with 1 M KCl or NaCl. The accumulated betaine was not metabolized further; transport was relatively specific for betaine and was dependent on an energy source. Other lactobacilli, more osmosensitive than strain 3532, including L. acidophilus strain E4356, L. bulgaricus 8144, and L. delbrueckii 9649, showed lower betaine transport rates in response to an osmotic challenge than L. acidophilus 3532. Experiments with chloramphenicol-treated L. acidophilus 3532 cells indicated that the transport system was not induced but appeared to be activated by an increase in osmotic pressure.     

Small Multidrug Resistance Protein EmrE Reduces Host pH and Osmotic Tolerance to Metabolic Quaternary Cation Osmoprotectants

The small multidrug resistance (SMR) transporter protein EmrE in Escherichia coli is known to confer resistance to toxic antiseptics classified as quaternary cation compounds (QCCs). Naturally derived QCCs synthesized during metabolic activities often act as osmoprotectants, such as betaine and choline, and participate in osmotic homoestasis. The goal of this study was to determine if EmrE proteins transport biological QCC-based osmoprotectants. Plasmid-encoded copies of E. coli emrE and the inactive variant emrE-E14C (emrE with the E→C change at position 14) were expressed in various E. coli strains grown in either rich or minimal media at various pHs (5 to 9) and under hypersaline (0.5 to 1.0 M NaCl and KCl) conditions to identify changes in growth phenotypes induced by osmoprotectant transport. The results demonstrated that emrE expression reduced pH tolerance of E. coli strains at or above neutral pH and when grown in hypersaline media at or above NaCl or KCl concentrations of 0.75 M. Hypersaline growth conditions were used to screen QCC osmoprotectants betaine, choline, l-carnitine, l-lysine, l-proline, and l-arginine. The study identified that betaine and choline are natural QCC substrates of EmrE.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Role for Glycine Betaine Transport in Vibrio cholerae Osmoadaptation and Biofilm Formation within Microbial Communities

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Conversion of quaternary ammonium compounds in the whole plant system of the halophyte Suaeda altissima

The total content of quaternary ammonium compounds (QAC) and the choline content in roots, leaves, and xylem exudates of the halophyte Suaeda altissima (L.) Pall. were determined after growing plants at various NaCl concentrations in the nutrient solution (1, 50, 100, and 250 mM). Based on the results obtained, the content of glycine betaine in organs and xylem exudates of S. altissima was estimated as the difference between the total content of QAC and the choline content. In roots choline accounted for the largest portion of QAC (from 69 to 96% at various NaCl concentrations in nutrient media), whereas in leaves it contributed only 12–23%. The contribution of choline to QAC content in the xylem exudates was 84–90%. It is concluded that choline in S. altissima is mainly synthesized in roots and is delivered with the ascending water flow to leaves where it is utilized as a substrate for glycine betaine synthesis. The content of glycine betaine in leaves increased with elevation of NaCl concentration in the nutrient solution, thus contributing appreciably to the maintenance of osmotic balance in the cytoplasm of S. altissima at high salinity.     

Occurrence of Choline and Glycine Betaine Uptake and Metabolism in the Family Rhizobiaceae and Their Roles in Osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.     

High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a K_i of approximately 4 μM. Uptake studies with [1,2-dimethyl-¹⁴C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a K_m value of 4 ± 1 μM and a maximum rate of transport (V_max) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance ¹³C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and γ-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83–90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.     

Glycine betaine, carnitine, and choline enhance salinity tolerance and prevent the accumulation of sodium to a level inhibiting growth of Tetragenococcus halophila

Natural-abundance (13)C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na(+) content and confer a much higher salt tolerance to T. halophila.     

Effect of glycine betaine on osmoadaptation of Propionibacterium acidipropionici cultivated in elevated osmolarities

The sensitivity of industrial strains Acetobacter aceti, Gluconobacter frateurii, and Propionibacterium acidipropionici to osmotic stress was studied. Growth of A. aceti and G. frateurii was totally inhibited at 0.4 M NaCl concentration, but P. acidipropionici was able to grow on a medium containing 1.2 M NaCl. Addition of glycine betaine to the medium had no detectable osmoprotective effect on A. aceti and G. frateurii cultivations in elevated NaCl concentrations, but it enabled cells of P. acidipropionici to achieve faster the maximum specific growth rate after the prolonged lag phase and therefore to gain faster the final biomass and product concentrations. The final concentrations of biomass and product of P. acidipropionici were the same as for the cultivations of the bacterium without NaCl and glycine betaine present in the medium. Intracellular accumulation of glycine betaine was detected in P. acidipropionici cells cultivated in the medium containing glycine betaine. The amount accumulated increased with NaCl concentration, suggesting that glycine betaine plays an important role in the osmoadaptation. Received: 31 March 2000 / Received revision: 22 May 2000 / Accepted: 3 June 2000     

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.